Method and device for processing a biological fluid for analyte determination

ABSTRACT

The present invention refers to a method of processing a biological fluid which comprises cellular components by heat treatment. The method is particularly useful for preparing biological samples for analyte detection. Further, the invention refers to a processed biological fluid comprising substantially quantitatively disintegrated cellular components.

The present invention refers to a method and device for processing abiological fluid which comprises cellular components by heat treatment.The method is particularly useful for preparing biological samples foranalyte detection. Further, the invention refers to a processedbiological fluid comprising substantially quantitatively disintegratedcellular components.

The determination of analytes in samples from biological fluids oftenrequires complicated and tedious pretreatment procedures in order toremove cellular components from the fluid sample. For example, wholeblood contains components, namely erythrocytes, leukocytes andthrombocytes. In order to determine analytes in a blood sample, thesecellular components often have to be removed by pre-treatment proceduressuch as centrifugation, filtration, sedimentation and/or homogenized bylysis using chemical reagents or mechanical treatment. These procedures,however, are often difficult to integrate into an automated test format.This holds also for a situation in which the target analytes are presentin the cellular components, e.g. immuno-suppressive drugs inerythrocytes. In this case, the cellular components either are isolatedor enriched by centrifugation and/or filtration prior to the addition ofa lysis reagent or they are denatured by addition of a denaturing agentto the original sample, for example a mixture of ZnSO₄ and acetonitrile,or the original sample is treated with temperatures of −20 to −170° C.

An object of the present invention was to provide an improved method forprocessing biological fluids which does not have the disadvantagesassociated with prior art procedures.

A first aspect of the present invention is a method of processing abiological fluid which comprises cellular components,

wherein the fluid is subjected to a heat treatment under conditions

-   (i) to provide substantially quantitative disintegration of said    cellular components and-   (ii) not to cause substantial sedimentation, precipitation,    denaturation, agglutination and gelation of fluid components.

A further aspect of the present invention is a processed biologicalfluid comprising substantially quantitatively disintegrated cellularcomponents which is substantially free from sedimentation,precipitation, denaturation, agglutination and gelation products.

Still a further aspect of the present invention is a method ofdetermining an analyte in a biological fluid sample, wherein thebiological fluid is processed as described above and the analyte isdetermined in the processed biological fluid.

Still a further aspect of the present invention is a device forprocessing a biological fluid, which comprises cellular componentscomprising:

-   (a) a fluid introduction port,-   (b) a fluid processing conduit, which is at least partially    heatable,-   (c) a heating element for heating a predetermined part of the fluid    processing conduit,-   (d) a fluid transportation element, e.g. a pumping element,-   (e) a control element for controlling the heating of the fluid under    conditions    -   (i) to provide substantially quantitative disintegration of said        cellular components and    -   (ii) not to cause substantial sedimentation, precipitation,        denaturation, agglutination and gelation of fluid components.-   (f) optionally a cleaning element and-   (g) optionally a sample analyzing element.

Surprisingly, the present inventor has found that a completedisintegration of cellular components, preferably cells or cell clustersfrom higher organisms, more preferably animal cells such as mammaliancells including human cells, and most preferably blood cells such aserythrocytes, leukocytes and/or thrombocytes in biological samples maybe achieved by heat treatment under predetermined conditions of time andtemperature. By means of this heat treatment, the cellular componentscontained in a biological fluid are disintegrated without substantialsedimentation, precipitation, denaturation, agglutination and/orgelation of fluid components.

The heat treatment may be carried out at a temperature of from 60-90°C., preferably from 60-75° C. and more preferably from 65-70° C. Theheat treatment is carried out for a time sufficient to achieve a desireddisintegration at the chosen treatment temperature. Preferably, the heattreatment is carried out for a time of 5 sec to 1 min, more preferablyfrom 10 sec to 40 sec. Especially preferred is a temperature of 70° C.for 30-40, e.g. 35 sec. The heat treatment may be carried out in anysuitable container, e.g. a glass-capillary (55×0.5 mm inner diameter).

In Table 1, suitable conditions for the heat treatment of a whole bloodsample with a given erythrocyte count, optionally supplemented withorganic solvents and/or plasma of blood group AB are shown. Thesetemperature/time conditions are defined by an upper time limit(indicated as value tmax) at a given temperature and by a lower timelimit (indicated as value tmin) at a given temperature. If the heattreatment is carried out for a time period longer than the tmax value,gelation occurs. If the heat treatment period is shorter than the tminvalue, only incomplete disintegration occurs. If other fluids, e.g.organic solvents and/or aqueous fluids, e.g. plasma, are added to thesample before heat treatment, the values of tmax and tmin may vary asshown in Tables 2-6.

The gelation of the sample may be determined by an increase inviscosity. The completeness of disintegration may be determined by cellcounting, e.g. in a Neubauer counting chamber, by microscopic inspectionfor particular components and/or by lack of sediment formation aftercentrifugation. In this context, it should be noted that about 95% ofcellular blood components are represented by erythrocytes. Thus, thecell count in a blood sample is preferably determined by counting theerythrocytes.

By means of the present invention the cell count in the sample ispreferably reduced to 0.1% or less and more preferably to 0.01% or lessof the original value. For example, when subjecting a sample with 5×10⁶erythrocytes per μl to heat treatment, the cell count is preferablyreduced to 5×10³ cells or less per μl (cf. Example 1), more preferablyto 500 cells or less per μl. Most preferably, the sample is free fromdetectable cells. The absence of particular components may also bedetermined by light-microscopic observation, e.g. up to 100×magnification, and/or by centrifugation for 10 min at up to 3000 g,preferably at up to 7400 g.

The heat treatment may be carried out when the fluid is kept static,e.g. while the fluid is in a reaction vessel. In a preferred embodiment,however, particularly for automated operations, the heat treatment maybe carried out while the fluid is in flow, e.g. while the fluid ispassed through a conduit. The heat treatment in a flowing system may becarried out while passing the sample fluid through a heated conduit,e.g. a capillary conduit, preferably with an inner diameter of about0.1-0.8 mm, for example of about 0.5 mm with a predetermined flow rate,wherein the conduit has a predetermined length in order to achieve thedesired residence time within the heated conduit. The heating may becarried out by any suitable means and may comprise e.g. inductiveheating such as microwave treatment, for example as described in U.S.Pat. No. 6,605,454, convective heating, resistive heating and/or heatingby laser excitation.

The biological fluid may be a body fluid such as whole blood, urine,cerebrospinal fluid, saliva, lymph fluid etc. or fluid from a cellculture or any other biological fluid comprising cellular components,particularly fluids comprising blood cells. More preferably, thebiological fluid is whole blood, such as venous, arterial or capillaryblood, particularly anticoagulant-treated whole blood, e.g. EDTA-,citrate-, or heparin-treated whole blood. For example, a sample may betaken with an anticoagulant containing blood withdrawal device anddirectly subjected to further processing as described below.

The sample volume may be varied broadly, e.g. in the range of 1 nl ormore, preferably 10 nl or more and up to 1 ml. Thus, the method issuitable for miniaturized applications, e.g. microfluidic devices onchip format, nano LC-MS analysis etc.

The method of the present invention does not require sedimentationand/or precipitation and/or centrifugation steps and/or the addition ofchemical lysis and/or disintegration reagents. Thus, the heat treatmentis preferably carried out without previous removal and/or lysis ofcellular components. The method may be carried out in any suitabledevice, e.g. a single-use device or a reusable device. Preferably, themethod is an automated procedure, which may be carried out in anintegrated device, i.e. a device into which the fluid sample istransferred, optionally after mixing, e.g. with a further fluid, withoutpretreatment, particularly without removal and/or lysis of cellularcomponents. Within the device, the sample is preferably directlysubjected to the treatment without prior removal and/or a lysis ofcellular components. After treatment, subsequent steps, e.g. an analytedetermination may be carried out. Most preferably, the heat treatment iscarried out with a substantially native sample, e.g. a sample comprisingsubstantially intact cellular components such as whole blood.

The method of the present invention may include the addition of furtherfluid to the biological fluid before and/or after processing. Thefurther fluid may be an organic solvent, preferably in an amount of upto 20% (vol/vol), more preferably in an amount of up to 10% (vol/vol)based on the volume of the biological fluid. The organic solvent ispreferably selected from water-miscible solvents such as methanol,ethanol, acetonitrile, dimethylsulfoxide, and combinations thereof. Theaddition of organic solvents may have an influence on thetemperature/time conditions for the heat treatment as shown in Tables 2and 3.

Preferably, the further fluid does not substantially effect lysis ofcellular components. More preferably, the further fluid is an aqueousfluid, e.g. an aqueous buffer solution or a further biological fluid,preferably having a ionic strength corresponding to 0.5-1.4% NaCl, morepreferably 0.7-1.2% NaCl and most preferably about 0.9% NaCl.Preferably, the aqueous fluid is a biological fluid without cellularcomponents such as plasma. More preferably, the plasma is plasma of theblood group AB. The further aqueous fluid may comprise an organicsolvent, e.g. in an amount of up to 20% (vol/vol), preferably up to 10%(vol/vol) based on the volume of the second biological fluid such asmethanol, ethanol, acetonitrile, dimethylsulfoxide and/or combinationsthereof. The second biological fluid is preferably added in a volumeratio of 5:1 to 1:10 based on the volume of the first biological fluid.Preferably, the second fluid is added before the heat treatment. Theaddition of the second fluid may influence the suitable temperature/timeconditions for the heat treatment step as shown in Tables 4-6.Surprisingly, it was found that addition of AB plasma, optionally withan organic solvent, actually increases the suitable treatment time rangeat a given temperature.

The further fluid may be a standardisation and/or calibrator fluidcomprising a predetermined amount of at least one standardisationand/calibrator compound. The addition of standardisation and/orcalibrator compounds is particularly suitable if the heat-treatedbiological fluid is further analysed by means of chromatographic,spectrometric and/or spectroscopic methods. The standardisation and/orcalibrator compounds may be analyte analogues which contain stableisotopes such as ²H and/or ¹³C and thus may be detected by massspectrometry.

The method also may include the addition of a marker/staining compoundfor lipids, proteins, peptides, nucleic acids and carbohydrates to thebiological fluid before and/or after processing.

A further aspect of the present invention refers to a compositioncomprising AB plasma, an organic solvent in an amount of up to 20%(vol/vol), preferably up to 10% (vol/vol) based on the volume of theplasma, such as methanol, ethanol, acetonitrile and/or dimethylsulfoxideand a predetermined amount of at least one standardisation and/orcalibrator compound. The compound may be used as a standardisationand/or calibrator fluid, particularly in combination with the heattreatment procedure as described above.

Still a further aspect of the present invention refers to a processedbiological fluid which is obtainable as described above. The processedfluid represents a novel matrix which is particularly suitable forclinical testing. The processed fluid is stable at least 1 week,preferably at least 2 weeks at 4° C. and/or at least 1 day, preferablyat least 5 days, at 25° C. Thus, the handling of the processed fluid ismuch more uncomplicated compared to a non-treated whole blood sample.The term “stable” particularly means that sedimentation does not occur.

The processed biological fluid comprises substantially quantitativelydisintegrated cellular components such as components from blood cells.The processed fluid is substantially free from sedimentation,precipitation, denaturation, agglutination and/or gelation products. Ina preferred embodiment, the invention refers to a processed fluid whichsubstantially

-   (i) is free from particular components on microscopic observation    (e.g. 100× magnification);-   (ii) is free from sediment after centrifugation for 10 min. at up to    3000 g, preferably at up to 7400 g and/or-   (iii) is free from cells as determined in a cell counting chamber.

The processed fluid preferably has a ionic strength corresponding to0.5-1.4% NaCl, more preferably 0.7-1.2% NaCl and most preferably asubstantially physiological salt concentration. The processed fluid maybe free from added disintegration and/or lysis reagents and/ordetergents. On the other hand, the processed fluid may also compriseorganic solvents and/or added aqueous fluid such as plasma of bloodgroup AB as described above. Most preferably, the processed fluid isprocessed whole blood.

The present invention also refers to a method of determining an analytein a biological fluid sample which has been subjected to a heattreatment as described above. The analyte may be any analyte which maybe detected in biological fluids, e.g. a biological compound such as anucleic acid, a polypeptide, peptide, lipid, sugar, hormone, metabolite,etc. On the other hand, the analyte may be a non-biological compound,e.g. a pharmaceutical compound. In a preferred embodiment, the analyteis an immunosuppressive drug, such as cyclosporin, rapamycin ortacrolimus or related compounds.

The analyte determination in the processed fluid may be carried outaccording to any known method. For example, the analyte determinationmay be carried out according to chemical, biochemical and/orphysicochemical methods and may comprise a hybridization reaction, animmunological reaction, an enzymatic reaction, e.g. a nucleic acidamplification, a chromatographic analysis, a spectrometric analysis,such as a mass-spectrometric or a NMR analysis and/or a spectroscopicanalysis. In an especially preferred embodiment, the invention refers toa method of determining an immunosuppressive drug in a whole bloodsample, wherein the whole blood is processed by a heat treatment asdescribed above and the immunosuppressive drug is determined in theprocessed whole blood according to standard methods, e.g. bymass-spectrometric methods.

In a further preferred embodiment, the analyte is a clinical-chemicalparameter, e.g. a clinical-chemical parameter associated with an inbornmetabolic disorder, e.g. phenylketonuria. In this embodiment, the sampleis preferably a capillary blood sample which may be obtained fromnewborns.

In a still further preferred embodiment, the method is suitable forprocessing blood samples from non-human animals, preferably mice, guineapigs and rats. For example, the samples may be taken by automatedsystems and directly processed as described above. A preferred automatedsystem is the Accu Sampler® from DiLab®.

A device of the present invention may comprise a fluid introductionport, where a sample of a biological fluid may e.g. be injected into thedevice. The fluid is transported within the device by a fluidtransportation element, e.g. a pumping element. Further, the device maycomprise a fluid processing conduit which is at least partiallyheatable. The heatable part of the fluid processing conduit may be anintegral part of the device or removably attached to the device. Thefluid processing conduit has preferably an inner diameter of about0.1-0.8 mm. In order to achieve a desired residence time within heatableportion of the conduit a predetermined flow rate of the biological fluidmay be adjusted. The heating element may be any suitable heatingelement, e.g. an element for inductive heating, an element forconvective heating, an element for resistive heating and/or an elementfor heating by laser excitation. For example, the heating element may bea heating coil wrapped around a predetermined part of the fluidprocessing conduit or a microwave emitter. The control element providescontrol of the sample processing, particularly the heating of the fluid,e.g. by controlling the heating intensity and/or time and/or the fluidflow rate in the heatable part of the fluid processing conduit.

The device may optionally comprise a cleaning element which is suitablefor cleaning the fluid processing conduit or at least a part thereof.For example, the cleaning element is adapted for carrying out a cleaningof the fluid processing conduit or a part thereof after a predeterminednumber of biological fluid processing cycles. Preferably, the cleaningcomprises passing a cleaning fluid through the fluid processing conduitor a part thereof. The cleaning fluid is capable of removing biological,e.g. proteinaceous residues in the processing conduit. Preferably, thecleaning fluid is an alkaline hypochlorite solution, e.g. an alkalineNaOCl solution. The cleaning may involve flushing of the fluidprocessing conduit or a part thereof with the cleaning fluid, whereinthe cleaning fluid is preferably at an elevated temperature, e.g. at atemperature of T≧60° C. The cleaning efficacy may be controlled bymonitoring the presence of biological materials in the fluid processingconduit or a part thereof after a cleaning procedure. The monitoringpreferably comprises a photometric detection of biological material,e.g. proteinaceous material. The detection may be carried out bydetermining biological materials, which have been solubilised/hydrolysedby the cleaning fluid, preferably in an online detection mode.Biological materials may be determined by a suitable colour reaction,e.g. the OPA reaction wherein O-phthaldialdehyde andN,N-dimethyl-2-mercaptoethyl-ammonium chloride may react with primaryamine compounds, e.g. proteins or hydrolysis products, under alkalineconditions (e.g. 0.1 mol/l Na₂B₄O₇ pH 9.3) to an1-alkylthio-2-alkylisoindole, which may be photometrically detected at340 nm.

Further, the device optionally comprises a sample analysing element. Thesample analysing element may be any element which is suitable foranalyte detection in a biological sample. Preferably, the sampleanalysing element comprises a chromatographic element, e.g. an HPLCelement, an extraction element, e.g. a Solid Phase Extraction (SPE)element, a spectrometric element, e.g. a mass-spectrometric or NMRelement, a spectroscopic element, an enzymatic and/or immunoassayelement and/or a hybridization assay element.

Finally, the device may comprise a processor unit which may transferdata to and/or receive data from a remote control unit. The datatransfer may occur online, e.g. by wireless transfer such as viaGSM/GPRS/3G data transfer. The remote control unit may be adapted toauthorise fluid processing for a respective device, e.g. after paymentfor carrying out a predetermined number of fluid processing procedureshas been received (i.e. pay-per-process).

Further, the present invention is explained in more detail by thefollowing examples.

EXAMPLE 1 Heat Treatment of Blood Samples (Static System)

A glass-capillary (55 mm length×0.5 mm inner diameter) is filled withabout 10 μl of a blood sample (erythrocytes: 5.18×10⁶/μl; hemoglobin:17.5 g/dl; hematocrite 50.1%), sealed at one end with plasticine andheated in a thermostated water-bath at a given temperature for a giventime. At the end of the heating process the glass-capillary isimmediately immersed in an ice-bath (4° C.). The plasticine sealing iscut off, the glass-capillary is emptied and the treated blood sample isfurther investigated for gelation and completeness of blood celldisintegration. The parameter t_(max) is defined as the heating time[sec] at which gelation of the sample occurs minus 1 second. Theparameter t_(min) is defined as the minimal heating time at which noerythrocytes are detected using a Neubauer counting chamber.

EXAMPLE 2 Erythrocyte Count

To 10 μl of whole blood or treated blood 990 μl of Hayemsch-solution(Merck KGaA, Darmstadt, Germany) are added. The mixture is vortexed andan aliquot is introduced into a Neubauer counting chamber. Theerythrocytes present in 5 defined squares are counted using a microscopeand a magnification of 100.

Calculation:

${{{Erythrocytes}/\mu}\; l\mspace{14mu}({sample})} = \frac{{{Number}\mspace{14mu}{of}\mspace{20mu}{erythrocytes}\mspace{14mu}{counted}\mspace{14mu} \times \mspace{14mu} 100}\mspace{11mu}}{0.2\mspace{14mu}{mm}^{2}\mspace{14mu} \times \mspace{14mu} 0.1\mspace{14mu}{mm}}$

The results are shown in the following Tables 1-6.

TABLE 1 Heat treatment of whole blood (erythrocytes: 5.18 × 10⁶/μl;hemoglobin: 17.5 g/dl; hematocrite 50.1%). Temperature [° C.] t_(max)[sec] t_(min) [sec] 90 3 3 85 5 4 80 9 5 75 23 9 70 48 21 65 242 33 60802 349

TABLE 2 Heat-treatment of whole blood containing 5 vol % of methanolTemperature [° C.] t_(max) [sec] t_(min) [sec] 80 6 6 75 13 7 70 42 1165 169 18 60 646 21

TABLE 3 Heat-treatment of whole blood containing 5 vol % of acetonitrileTemperature [° C.] t_(max) [sec] t_(min) [sec] 80 6 5 75 10 9 70 21 1065 49 11 60 184 32

TABLE 4 Heat-treatment of a 1:1 mixture of whole blood and AB-plasmaTemperature [° C.] t_(max) [sec] t_(min) [sec] 80 15 8 75 31 9 70 65 4165 412 66

TABLE 5 Heat-treatment of a 1:1 mixture of whole blood and AB-plasmacontaining 5 vol % of methanol Temperature [° C.] t_(max) [sec] t_(min)[sec] 80 5 3 75 13 3 70 38 3 65 126 8 60 693 28

TABLE 6 Heat-treatment of a 1:1 mixture of whole blood and AB-plasmacontaining 2.5, 5 or 10 vol % acetonitrile t_(min) [sec] t_(max) [sec]acetonitrile Temperature acetonitrile vol % vol % [° C.] 2.5 5 10 2.5 510 80 11 5 3 5 4 1 75 23 11 4 3 2 1 70 41 24 5 4 5 1 65 171 53 17 10 121 60 753 281 35 11 33 2

EXAMPLE 3 Heat Treatment of a Blood Sample (Flow System)

For the treatment of a blood sample (e.g. 10 μl) using a heatedstainless-steel capillary with the dimension of 0.5 mm internal diameterand 300 mm in length, the heating time at a given temperature can bepreadjusted via the flow-rate of a given test fluid such as 0.9 vol %NaCl solution.

For a temperature of 75° C. the minimal capillary retention time t_(min)of 9 sec (cf. Example 1, Table 1) is reached with a flow-rate of 466μl/min and the maximal capillary retention time t_(max) of 23 sec. (cf.Example 1, Table 1) is reached with a flow-rate of 183 μl/min. Thus, apreferred flow-rate for such a sample size and capillary configurationwould be within these boundaries, e.g. amounting to approximately 325μl/min. This flow-rate is also optimal for electrospray-ionisation inmass spectrometry.

Calculation:

${{Flow}\mspace{14mu}{{rate}\mspace{14mu}\left\lbrack {{\mu l}\text{/}\min} \right\rbrack}} = {\frac{{{Volume}\mspace{14mu}{{Capillary}\mspace{14mu}\lbrack{\mu l}\rbrack}} + {{Volume}\mspace{14mu}{{Sample}\mspace{14mu}\left\lbrack {\mu\; l} \right\rbrack}}}{{t_{\min}\left( {{or}\mspace{14mu} t_{\max}} \right)}\mspace{14mu}\left\lbrack \sec \right\rbrack} \times 60}$

1. A method of conducting a heat treatment of a biological fluid whichcomprises cellular components, comprising: heating the biological fluidto a temperature of from 60-90° C.; and maintaining the temperature ofthe biological fluid for a period of from 10 seconds to 40 seconds;wherein the fluid is subjected to the heat treatment under conditions(i) to provide substantially quantitative disintegration of saidcellular components and (ii) not to cause substantial sedimentation,precipitation, denaturation, agglutination and gelation of fluidcomponents.
 2. The method of claim 1 wherein the heat treatment iscarried out at a temperature of from 60-75° C.
 3. The method of claim 1wherein the heat treatment is carried out while the fluid is static. 4.The method of claim 1 wherein the heat treatment is carried out using aheating method selected from the group consisting of inductive heating,convective heating, resistive heating and heating by laser excitation.5. The method of claim 1 wherein the biological fluid is a body fluid ora cell culture fluid.
 6. The method of claim 1 which does not include(i) a sedimentation and/or precipitation step and/or centrifugation stepand/or (ii) an addition of chemical disintegration and/or lysisreagents.
 7. The method of claim 1 which is carried out as an automatedprocedure.
 8. The method of claim 1 further comprising adding a furtherfluid to the biological fluid before and/or after said heat treatment.9. The method of claim 1 wherein the biological fluid is whole blood.10. A processed biological fluid obtained by the method of claim
 1. 11.The processed fluid of claim 10 comprising substantially quantitativelydisintegrated cellular components which is substantially free fromsedimentation, precipitation, denaturation, agglutination and gelationproducts.
 12. The processed fluid of claim 10 which substantially (i) isfree from particulate components on microscopic observation; (ii) isfree from sediment after centrifugation; and/or (iii) is free from cellsas determined in a cell counting chamber.
 13. A method of conducting aheat treatment of a biological fluid which comprises cellularcomponents, comprising: heating the biological fluid to a temperature offrom 60-90° C.; and maintaining the temperature of the biological fluidfor a period of from 5 seconds to 1 minute; wherein the fluid issubjected to the heat treatment under conditions (i) to providesubstantially quantitative disintegration of said cellular componentsand (ii) not to cause substantial sedimentation, precipitation,denaturation, agglutination and gelation of fluid components, whereinthe heat treatment is carried out while the fluid is in flow.
 14. Amethod of conducting a heat treatment of a biological fluid whichcomprises cellular components, comprising: heating the biological fluidto a temperature of from 60-90° C.; and maintaining the temperature ofthe biological fluid for a period of from 5 seconds to 1 minute; whereinthe fluid is subjected to the heat treatment under conditions (i) toprovide substantially quantitative disintegration of said cellularcomponents and (ii) not to cause substantial sedimentation,precipitation, denaturation, agglutination and gelation of fluidcomponents, wherein the method further comprises adding a further fluidto the biological fluid before and/or after said heat treatment, and thefurther fluid comprises an organic solvent in an amount of up to 20%(vol/vol) based on the volume of the biological fluid, and wherein thefurther fluid is selected from the group consisting of methanol,ethanol, acetonitrile and dimethylsulfoxide.
 15. A method of conductinga heat treatment of a biological fluid which comprises cellularcomponents, comprising: heating the biological fluid to a temperature offrom 60-90° C.; and maintaining the temperature of the biological fluidfor a period of from 5 seconds to 1 minute; wherein the fluid issubjected to the heat treatment under conditions (i) to providesubstantially quantitative disintegration of said cellular componentsand (ii) not to cause substantial sedimentation, precipitation,denaturation, agglutination and gelation of fluid components, whereinthe method further comprises adding a further fluid to the biologicalfluid before and/or after said heat treatment, and the further fluidcomprises an aqueous fluid without cellular components, wherein theaqueous fluid optionally further comprises an organic solvent in anamount of up to 20% (vol/vol) based on the volume of the aqueous fluid.16. The method of claim 15 wherein the aqueous fluid without cellularcomponents is plasma.
 17. A method of conducting a heat treatment of abiological fluid which comprises cellular components, comprising:heating the biological fluid to a temperature of from 60-90° C.; andmaintaining the temperature of the biological fluid for a period of from5 seconds to 1 minute; wherein the fluid is subjected to the heattreatment under conditions (i) to provide substantially quantitativedisintegration of said cellular components and (ii) not to causesubstantial sedimentation, precipitation, denaturation, agglutinationand gelation of fluid components, wherein the method further comprisesadding a further fluid to the biological fluid before and/or after saidheat treatment, and the further fluid is a standardization and/orcalibrator fluid comprising a predetermined amount of at least onestandardization and/or calibrator compound.
 18. A processed biologicalfluid obtained by conducting a heat treatment of a biological fluidwhich comprises cellular components, comprising: heating the biologicalfluid to a temperature of from 60-90° C.; and maintaining thetemperature of the biological fluid for a period of from 5 seconds to 1minute; wherein the fluid is subjected to the heat treatment underconditions (i) to provide substantially quantitative disintegration ofsaid cellular components and (ii) not to cause substantialsedimentation, precipitation, denaturation, agglutination and gelationof fluid components, wherein the processed biological fluid has asubstantially physiological salt concentration.
 19. A processedbiological fluid obtained by conducting a heat treatment of a biologicalfluid which comprises cellular components, comprising: heating thebiological fluid to a temperature of from 60-90° C.; and maintaining thetemperature of the biological fluid for a period of from 5 seconds to 1minute; wherein the fluid is subjected to the heat treatment underconditions (i) to provide substantially quantitative disintegration ofsaid cellular components and (ii) not to cause substantialsedimentation, precipitation, denaturation, agglutination and gelationof fluid components, wherein the processed biological fluid is free fromadded reagents.
 20. A processed biological fluid obtained by conductinga heat treatment of a biological fluid which comprises cellularcomponents, comprising: heating the biological fluid to a temperature offrom 60-90° C.; and maintaining the temperature of the biological fluidfor a period of from 5 seconds to 1 minute; wherein the fluid issubjected to the heat treatment under conditions (i) to providesubstantially quantitative disintegration of said cellular componentsand (ii) not to cause substantial sedimentation, precipitation,denaturation, agglutination and gelation of fluid components, whereinthe processed biological fluid is cell-disintegrated blood.
 21. A methodof conducting a heat treatment of a biological fluid which comprisescellular components, wherein the biological fluid comprises whole blood,wherein the fluid is subjected to the heat treatment under conditions(i) that provide substantially quantitative disintegration of saidcellular components; and (ii) that do not cause substantialsedimentation, precipitation, denaturation, agglutination and gelationof fluid components, wherein the heat treatment is carried out underconditions of temperature and time defined by an upper time limit at agiven temperature and by a lower time limit at a given temperature, andwherein, when the heat treatment is carried out for a time period longerthat the upper time limit, gelation occurs, and wherein, when the heattreatment is carried out for a time period shorter than the lower timelimit, incomplete disintegration occurs, and wherein the cell count inthe biological fluid is reduced to 0.1% or less.
 22. The method of claim21 wherein the heat treatment is carried out at a temperature of from60-90° C.
 23. The method of claim 21 wherein the heat treatment iscarried out for a time of 5 sec to 1 minute.
 24. The method of claim 21wherein the heat treatment is carried out while the fluid is static. 25.The method of claim 21 wherein the heat treatment is carried out whilethe fluid is in flow.
 26. The method of claim 21 wherein the heattreatment is carried out using a heating method selected from the groupconsisting of inductive heating, convective heating, resistive heatingand heating by laser excitation.
 27. The method of claim 21 wherein thebiological fluid comprises anti-coagulant treated whole blood.
 28. Themethod of claim 21 which does not include (i) a sedimentation and/orprecipitation step and/or centrifugation step and/or (ii) an addition ofchemical disintegration and/or lysis reagents.
 29. The method of claim21 which is carried out as an automated procedure.
 30. The method ofclaim 21 further comprising adding a further fluid to the biologicalfluid before and/or after said heat treatment.
 31. The method of claim30 wherein the further fluid is an organic solvent in an amount of up to20% (vol/vol) based on the volume of the biological fluid, and whereinthe further fluid is selected from the group consisting of methanol,ethanol, acetonitrile and dimethylsulfoxide.
 32. The method of claim 30wherein the further fluid is a standardization and/or calibrator fluidcomprising a predetermined amount of at least one standardization and/orcalibrator compound.
 33. The method of claim 30 wherein the furtherfluid is an aqueous fluid without cellular components, wherein theaqueous fluid optionally further comprises an organic solvent in anamount of up to 20% (vol/vol) based on the volume of the aqueous fluid.34. The method of claim 33 wherein the aqueous fluid without cellularcomponents is plasma.
 35. A processed biological fluid obtained by themethod of claim
 21. 36. The processed fluid of claim 35 comprisingsubstantially quantitatively disintegrated cellular components which issubstantially free from sedimentation, precipitation, denaturation,agglutination and gelation products.
 37. The processed fluid of claim 35which substantially (i) is free from particulate components onmicroscopic observation; (ii) is free from sediment aftercentrifugation; and/or (iii) is free from cells as determined in a cellcounting chamber.
 38. The processed fluid of claim 35 which has asubstantially physiological salt concentration.
 39. The processed fluidof claim 35 which is free from added reagents.
 40. The processed fluidof claim 35 which is cell-disintegrated blood.